Fluorescence observation apparatus and fluorescence observation method

ABSTRACT

A fluorescence observation apparatus according to an embodiment of the present technology includes a stage, an excitation section, and a spectroscopic imaging section. The stage is capable of supporting a fluorescently stained pathological specimen. The excitation section irradiates the pathological specimen on the stage with a plurality of line illuminations of different wavelengths, the plurality of line illuminations being a plurality of line illuminations situated on different axes and parallel to a certain-axis direction. The spectroscopic imaging section includes at least one imaging device capable of separately receiving pieces of fluorescence respectively excited with the plurality of line illuminations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 371 as a U.S.National Stage Entry of International Application No. PCT/JP2019/021509,filed in the Japanese Patent Office as a Receiving Office on May 30,2019, which claims priority to Japanese Patent Application NumberJP2018-103507, filed in the Japanese Patent Office on May 30, 2018, eachof which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present technology relates to, for example, a fluorescenceobservation apparatus and a fluorescence observation method that areused to perform diagnosis using a pathological image.

BACKGROUND ART

A pathological-image diagnosis using fluorescence staining has beenproposed as a highly quantitative and polychromatic method (refer to,for example, Patent Literature 1). Compared with colored staining, thefluorescent approach has the advantage that it is possible to easilyperform multiplexing and to obtain detailed diagnostic information. Influorescence imaging other than fluorescence imaging used forpathological diagnosis, an increase in the number of colors makes itpossible to examine, at a time, various antigens expressed in a sample.

In general fluorography, excitation light of an absorption wavelength(an excitation wavelength) of a dye is irradiated, and a dye spectrumemitted by the irradiation is selectively incorporated using a bandpassfilter. When there exists a plurality of colors, the absorptionwavelength (the excitation wavelength) varies depending on the dye.Thus, a method for performing image-capturing while switching a filterfor each dye, is adopted. However, a plurality of dyes is excited at asingle excitation wavelength when multicolor staining is performed,since an absorption spectrum and an emission spectrum of a dye are bothbroad and overlap. Further, fluorescence of an adjacent dye is leaked toa bandpass filter, and this results in color mixture.

On the other hand, a method for performing image-capturing whileswitching a wavelength of excitation light and a wavelength of detectedfluorescence in a time-division manner, is known (for example,Non-Patent Literature 1). However, this method has a problem in whichthe image-capturing time is linearly increased as the number of colorsis increased.

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent No. 4452850

Non-Patent Literature

-   Non-Patent Literature 1: Edward C. Stack, “Multiplexed    immunohistochemistry, imaging, and quantitation: A review, with an    assessment of Tyramide signal amplification, multispectral imaging    and multiplex analysis”, Methods 70 (2014) 46-58

DISCLOSURE OF INVENTION Technical Problem

In view of the circumstances described above, it is an object of thepresent technology to provide a fluorescence observation apparatus and afluorescence observation method that make it possible to suppress anincrease in the image-capturing time that is associated with an increasein the number of observation-target dyes.

Solution to Problem

A fluorescence observation apparatus according to an embodiment of thepresent technology includes a stage, an excitation section, and aspectroscopic imaging section.

The stage is capable of supporting a fluorescently stained pathologicalspecimen.

The excitation section irradiates the pathological specimen on the stagewith a plurality of line illuminations of different wavelengths, theplurality of line illuminations being a plurality of line illuminationssituated on different axes and parallel to a certain-axis direction.

The spectroscopic imaging section includes at least one imaging devicecapable of separately receiving pieces of fluorescence respectivelyexcited with the plurality of line illuminations.

This makes it possible to suppress an increase in the image-capturingtime that is associated with an increase in the number ofobservation-target dyes.

The excitation section may be configured to irradiate, onto thepathological specimen and as the plurality of line illuminations, aplurality of line illuminations each having different wavelengths incombination.

The spectroscopic imaging section may further include a wavelengthdispersion element that separates each of the pieces of fluorescencerespectively excited with the plurality of line illuminations.

The spectroscopic imaging section may further include an observationslit including a plurality of slit portions, each of the pieces offluorescence respectively excited with the plurality of lineilluminations being allowed to pass through a corresponding one of theplurality of slit portions.

The fluorescence observation apparatus may further include a scanningmechanism that scans the plurality of line illuminations over the stagein a direction orthogonal to the certain-axis direction.

The fluorescence observation apparatus may further include a processingunit that includes a storage that stores therein spectroscopic data thatindicates a correlation between a wavelength of each of the plurality ofline illuminations and fluorescence received by the imaging device.

The processing unit may further include an image formation section thatforms a fluorescence image of the pathological specimen on the basis ofthe spectroscopic data stored in the storage and an interval between theplurality of line illuminations.

The image formation section may be configured to form, as thefluorescence image, an image in which coordinates detected by theimaging device have been corrected using a value corresponding to theinterval between the plurality of line illuminations.

The processing unit may further include a data calibration section thatcalibrates spectroscopic data stored in the storage.

The storage may store therein standard spectra in advance, the standardspectra being a standard spectrum of autofluorescence related to thepathological specimen and a standard spectrum of only a dye that stainsthe pathological specimen, and the image formation section may beconfigured to output a component distribution of the spectroscopic dataon the basis of the standard spectrum of the autofluorescence and thestandard spectrum of only the dye.

The imaging device may include a plurality of imaging devices eachcapable of receiving the fluorescence passing through the observationslit.

The fluorescence observation apparatus may further include anon-fluorescence observation section that includes a light source thatilluminates the pathological specimen on the stage, and an imagingsection that acquires a non-fluorescence image of the pathologicalspecimen.

The fluorescence observation apparatus may further include a displaysection that displays fluorescence spectra separately for each of theplurality of line illuminations, the fluorescence spectra beingrespectively excited with the plurality of line illuminations.

The display section may include an operation region in which awavelength and output of each of the plurality of line illuminations areallowed to be set.

The display section may include a display region used to display adetection wavelength range for the fluorescence spectrum.

A fluorescence observation method according to an embodiment of thepresent technology includes:

irradiating a pathological specimen on a stage with a plurality of lineilluminations of different wavelengths, the plurality of lineilluminations being a plurality of line illuminations situated ondifferent axes and parallel to a certain-axis direction; and

separately receiving pieces of fluorescence respectively excited withthe plurality of line illuminations.

The fluorescence observation method may further include scanning theplurality of line illuminations over the stage in a direction orthogonalto the certain-axis direction.

A plurality of line illuminations each having different wavelengths incombination may be used as the plurality of line illuminations.

Advantageous Effects of Invention

As described above, the present technology makes it possible to suppressan increase in the image-capturing time that is associated with anincrease in the number of observation-target dyes.

Note that the effect described here is not necessarily limitative, andany of the effects described in the present disclosure may be provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic block diagram of a fluorescence observationapparatus according to an embodiment of the present technology.

FIG. 2 illustrates an example of an optical system in the fluorescenceobservation apparatus.

FIG. 3 schematically illustrates a pathological specimen of anobservation target.

FIG. 4 schematically illustrates how a line illumination is irradiatedonto the observation target.

FIG. 5 is a diagram describing a method for acquiring spectroscopic datawhen an imaging device in the fluorescence observation apparatusincludes a single image sensor.

FIG. 6 illustrates the wavelength characteristics of the spectroscopicdata acquired in FIG. 5 .

FIG. 7 is a diagram describing the method for acquiring spectroscopicdata when the imaging device includes a plurality of image sensors.

FIG. 8 is a conceptual diagram describing a method for scanning the lineillumination irradiated onto the observation target.

FIG. 9 is a conceptual diagram describing pieces of three-dimensionaldata (X, Y, λ) acquired using a plurality of line illuminations.

FIG. 10 illustrates configuration examples of a wavelength of anexcitation section in the fluorescence observation apparatus.

FIG. 11 schematically illustrates another configuration example of aspectroscopic imaging section in the fluorescence observation apparatus.

FIG. 12 is a flowchart illustrating an example of a procedure ofprocessing performed in a processing unit in the fluorescenceobservation apparatus.

FIG. 13 is a diagram describing a screen of a display section in thefluorescence observation apparatus.

FIG. 14 illustrates an example of a screen configuration of a settingregion in the display section, the setting region being a setting regionfor the excitation section.

FIG. 15 illustrates an example of a screen configuration of a detectionsetting region in the display section, the detection setting regionbeing a detection setting region for a fluorescence spectrum from a lineillumination.

FIG. 16 illustrates an example of a screen configuration of a detectionsetting region in the display section, the detection setting regionbeing a detection setting region for a fluorescence spectrum fromanother line illumination.

FIG. 17 is a schematic diagram conceptually illustrating a relationshipbetween fluorescence spectrum data and a fluorescence image displayed onthe display section.

FIG. 18 is a flowchart illustrating a modification of the procedure ofthe processing performed in the processing unit.

FIG. 19 is a flowchart illustrating another modification of theprocedure of the processing performed in the processing unit.

FIG. 20 is a schematic block diagram illustrating a modification of thefluorescence observation apparatus.

FIG. 21 is a schematic block diagram illustrating another modificationof the fluorescence observation apparatus.

MODE(S) FOR CARRYING OUT THE INVENTION

Embodiments according to the present technology will now be describedbelow with reference to the drawings.

FIG. 1 is a schematic block diagram of a fluorescence observationapparatus according to an embodiment of the present technology, and FIG.2 illustrates an example of an optical system in the fluorescenceobservation apparatus.

[Overall Configuration]

A fluorescence observation apparatus 100 of the present embodimentincludes an observation unit 1. The observation unit 1 includes anexcitation section 10 that irradiates a pathological specimen (apathological sample) with a plurality of line illuminations of differentwavelengths, the line illuminations being arranged parallel to eachother on different axes, a stage 20 that supports the pathologicalspecimen, and a spectroscopic imaging section 30 that acquires alinearly excited fluorescence spectrum (spectroscopic data) of thepathological specimen.

Here, being parallel to each other on different axes means that the lineilluminations are situated on different axis and parallel to each other.Being on different axes means not being on the same axis, and thedistance between the axes is not particularly limited. Being parallel toeach other is not limited to being accurately parallel to each other,and includes a state of being approximately parallel to each other. Forexample, there may be a deviation from a parallel state due to adistortion due to an optical system such as a lens, or due tomanufacturing tolerances, and such a state is also considered parallelto each other.

The fluorescence observation apparatus 100 further includes a processingunit 2. On the basis of a fluorescence spectrum of a pathologicalspecimen (hereinafter also referred to as a sample S) acquired by theobservation unit 1, the processing unit 2 typically forms an image ofthe pathological specimen or outputs a distribution of the fluorescencespectrum. Here, for example, the image refers to an image with colors ofRGB (red, green, and blue) that is obtained by performing conversionusing a component ratio of a dye included in a spectrum,autofluorescence from a sample, and the like, or using a waveform of thespectrum, or refers to a distribution of brightness in a specificwavelength range.

The excitation section 10 and the spectroscopic imaging section 30 areconnected to the stage 20 through an observation optical system 40 suchas an objective 44. The observation optical system 40 includes afunction of adjusting to an optimal focus using a focusing mechanism 60.A non-fluorescence observation section 70 used for dark field microscopyor bright field microscopy may be connected to the observation opticalsystem 40.

The fluorescence observation apparatus 100 may be connected to acontroller 80 that controls, for example, the excitation section(control of an LD and a shutter), an XY-stage that is a scanningmechanism, the spectroscopic imaging section (a camera), the focusingmechanism (a detector and a Z-stage), and the non-fluorescenceobservation section (a camera).

The excitation section 10 includes a plurality of light sources L1, L2,. . . that are capable of outputting pieces of light of a plurality ofexcitation wavelengths Ex1, Ex2, . . . . Each of the plurality of lightsources typically includes a light emitting diode (LED), a laser diode(LD), a mercury lamp, or the like. The piece of light of each of theplurality of light sources is changed to a line illumination, and theline illumination is irradiated onto the sample S on the stage 20.

The sample S is typically formed of a slide including an observationtarget Sa such as a tissue section illustrated in FIG. 3 . However, ofcourse, the sample S may be formed of something other than such a slide.The sample S (the observation target Sa) is stained with a plurality offluorescence dyes. The observation unit 1 magnifies the sample S to adesired magnification to observe the sample S. In an illuminationportion that is an enlarged portion A of FIG. 3 , a plurality of lineilluminations (two (Ex1 and Ex2) in the example) is arranged, and theimage-capturing areas R1 and R2 of the spectroscopic imaging section 30are arranged to overlap illumination areas of the respective lineilluminations, as illustrated in FIG. 4 . The two line illuminations Ex1and Ex2 are each parallel to a Z-axis direction, and are arranged awayfrom each other by a specified distance (Δy) in a Y-axis direction.

The image-capturing areas R1 and R2 respectively correspond to slitportions of an observation slit 31 (FIG. 2 ) in the spectroscopicimaging section 30. In other words, the same number of slit portions asthe number of line illuminations is arranged in the spectroscopicimaging section 30. Although the line width of the illumination islarger than the slit width in FIG. 4 , both the case in which the linewidth of the illumination is larger and the case in which the slit widthis larger may be acceptable. When the line width of the illumination islarger than the slit width, a margin used to align the excitationsection 10 with the spectroscopic imaging section 30 can be made larger.

The wavelength of the first line illumination Ex1 and the wavelength ofthe second line illumination Ex2 are different from each other. Piecesof linear fluorescence respectively excited with these lineilluminations Ex1 and Ex2 are observed in the spectroscopic imagingsection 30 through the observation optical system 40.

The spectroscopic imaging section 30 includes the observation slit 31including a plurality of slit portions, and at least one imaging device32 capable of separately receiving the pieces of fluorescence passingthrough the observation slit 31, in which the piece of fluorescenceexcited with each of the plurality of line illuminations is allowed topass through a corresponding one of the plurality of slit portions. Atwo-dimensional imager such as a charge coupled device (CCD) or acomplementary metal-oxide semiconductor (CMOS) is adopted as the imagingdevice 32. The arrangement of the observation slit 31 in a light pathmakes it possible to detect fluorescence spectra excited with therespective lines without an overlap of the fluorescence spectra.

The spectroscopic imaging section 30 acquires, from each of the lineilluminations Ex1 and Ex2, spectroscopic data (x, λ) of fluorescenceusing, as a wavelength channel, a pixel array situated in a certaindirection (for example, an orthogonal direction) of the imaging device32. The acquired spectroscopic data (x, λ) is recorded in the processingunit 2 in a state in which spectroscopic data is associated with anexcitation wavelength with which excitation is performed for thespectroscopic data.

The processing unit 2 may be implemented by hardware elements used in acomputer, such as a central processing unit (CPU), a random accessmemory (RAM), and a read only memory (ROM), and by necessary software.Instead of or in addition to the CPU, a programmable logic device (PLD)such as a field programmable gate array (FPGA), a digital signalprocessor (DSP), an application specific integrated circuit (ASIC), orthe like may be used.

The processing unit 2 includes a storage 21 that stores thereinspectroscopic data that indicates a correlation between wavelengths of aplurality of line illuminations Ex1 and Ex2 and fluorescence received bythe imaging device 32. A storage apparatus such as a nonvolatilesemiconductor memory or a hard disk drive is used as the storage 21, anda standard spectrum of autofluorescence related to the sample S and astandard spectrum of only a dye that stains the sample S are stored inthe storage 21 in advance. For example, the spectroscopic data (x, λ)received by the imaging device 32 is acquired as illustrated in FIGS. 5and 6 , and is stored in the storage 21. In the present embodiment, astorage that stores therein a standard spectrum of autofluorescence ofthe sample S and a standard spectrum of only a dye of the sample S, anda storage that stores therein spectroscopic data (a measurementspectrum) of the sample S that is acquired by the imaging device 32 areformed of the storage 21 in common. However, the storages are notlimited to this example, and they may be formed of separate storages.

FIGS. 5 and 6 are diagrams describing a method for acquiringspectroscopic data when the imaging device 32 includes a single imagesensor that receives every piece of fluorescence passing through theobservation slit 31. In this example, images of fluorescence spectra Fs1and Fs2 respectively excited with the line illuminations Ex1 and Ex2 arefinally formed on a light receiving surface of the imaging device 32through a spectroscopic optical system (described later) in a state inwhich the fluorescence spectra Fs1 and Fs2 are shifted from each otherby an amount proportional to Δy (refer to FIG. 4 ).

As illustrated in FIG. 5 , information obtained from the lineillumination Ex1 is recorded as Row_a and Row_b, and informationobtained from the line illumination Ex2 is recorded as Row_c and Row_d.Pieces of data in regions other than the regions of these pieces ofinformation are not read. Consequently, the frame rate of the imagingdevice 32 can be Row_full/(Row_b−Row_a+Row_d−Row_c) times faster thanwhen read is performed for a full frame.

As illustrated in FIG. 2 , a dichroic mirror 42 and a bandpass filter 45are inserted into a light path on the way such that excitation light(Ex1, Ex2) does not reach the imaging device 32. In this case, anintermittent portion IF is generated in the fluorescence spectrum Fs1 ofwhich an image is formed on the imaging device 32 (refer to FIGS. 5 and6 ). The frame rate can be further improved by excluding such anintermittent portion IF from a reading-target region.

As illustrated in FIG. 2 , the imaging device 32 may include a pluralityof imaging devices 32 a and 32 b each capable of receiving fluorescencepassing through the observation slit 31. In this case, the fluorescencespectra Fs1 and Fs2 respectively excited with the respective lineilluminations Ex1 and Ex2 are respectively acquired on the imagingdevices 32 a and 32 b, as illustrated in FIG. 7 , and are each stored inthe storage 21 in association with excitation light.

Each of the line illuminations Ex1 and Ex2 is not limited to having asingle wavelength, and may have a plurality of wavelengths. When each ofthe line illuminations Ex1 and Ex2 has a plurality of wavelengths,fluorescence excited at each of the plurality of wavelengths alsoincludes a plurality of spectra. In this case, the spectroscopic imagingsection 30 includes a wavelength dispersion element used to separate thefluorescence into spectra based on an excitation wavelength. Thewavelength dispersion element includes a diffraction grating, a prism,or the like, and is typically arranged in a light path between theobservation slit 31 and the imaging device 32.

The observation unit 1 further includes a scanning mechanism 50 thatscans the plurality of line illuminations Ex1 and Ex2 over the stage 20in the Y-axis direction, that is, in a direction in which the lineilluminations Ex1 and Ex2 are arranged. The use of the scanningmechanism 50 makes it possible to continuously record, in the Y-axisdirection, dye spectra (fluorescence spectra) that are excited atdifferent excitation wavelengths and are spatially separated from eachother by Δy on the sample S (the observation target Sa). In this case,an image-capturing region Rs is divided into multiple regions in anX-axis direction, for example, as illustrated in FIG. 8 , and anoperation is repeatedly performed, the operation including scanning thesample S in the Y-axis direction, subsequently moving the sample S inthe X-axis direction, and further performing scanning in the Y-axisdirection. It is possible to capture images of spectroscopic spectrafrom a sample in a single scan, the spectroscopic spectra being excitedat several excitation wavelengths.

The scanning mechanism 50 typically scans the stage 20 in the Y-axisdirection. However, the plurality of line illuminations Ex1 and Ex2 maybe scanned in the Y-axis direction using a galvanometer mirror arrangedin the optical system on the way. Finally, three-dimensional data (X, Y,λ) illustrated in FIG. 9 is acquired for each of the plurality of lineilluminations Ex1 and Ex2. Three-dimensional data based on one of theline illuminations Ex1 and Ex2 is data obtained by shifting thecoordinate by Δy from the coordinate of another of the lineilluminations Ex1 and Ex2 with respect to the Y-axis. Thus, correctionis performed on the basis of a value of Δy that is recorded in advance,or on the basis of a value of Δy that is calculated from output of theimaging device 32, and data obtained by the correction is output.

In the example described above, the number of line illuminations servingas excitation light is two, but it is not limited to this example. Thenumber of line illuminations may be three, four, or five or more.Further, each line illumination may also include a plurality ofexcitation wavelengths selected in order to minimize a degradation in acolor separation performance. Further, even when the number of lineillumination is one, it is possible to obtain a multicolor spectrum byusing an excitation light source including a plurality of excitationwavelengths and by recording the excitation wavelength in associationwith row data acquired by the imaging device, although the separationperformance provided by a plurality of line illuminations being parallelto each other on different axes, is not obtained. For example, theconfigurations illustrated in FIG. 10 may be adopted.

[Observation Unit]

Next, the observation unit 1 is described in detail with reference toFIG. 2 . Here, an example of the observation unit 1 to which aconfiguration example 2 of FIG. 10 is applied is described.

The excitation section 10 includes a plurality of (four in this example)excitation light sources L1, L2, L3, and L4. The excitation lightsources L1 to L4 are respectively formed of laser light sources thatrespectively output pieces of laser light of wavelengths of 405 nm, 488nm, 561 nm, and 645 nm.

The excitation section 10 further includes a plurality of collimatorlenses 11, a plurality of laser line filters 12, dichroic mirrors 13 a,13 b, and 13 c, a homogenizer 14, a condenser lens 15, and alight-entrance slit 16, in which each of the collimator lenses 11 andeach of the laser line filters 12 correspond to a respective one of theexcitation light sources L1 to L4.

Laser light emitted from the excitation light source L1 and laser lightemitted from the excitation light source L3 are each collimated by thecollimator lens 11, each pass through the laser line filter 12 used tonarrow its wavelength band, and are arranged on the same axis by thedichroic mirror 13 a. The two pieces of laser light on the same axis areeach further formed into a beam by the homogenizer 14 such as a fly eyelens and by the condenser lens 15 to become the line illumination Ex1.

Likewise, laser light emitted from the excitation light source L2 andlaser light emitted from the excitation light source L4 are arranged onthe same axis by the dichroic mirrors 13 b and 13 c to become a lineillumination that is the line illumination Ex2 situated on an axisdifferent from that of the line illumination Ex1. The line illuminationsEx1 and Ex2 form line illuminations on different axes (a primary image)in which the line illuminations Ex1 and Ex2 are situated away from eachother by Δy in the light-entrance slit 16 (slit conjugate) including aplurality of slit portions, in which each of the line illuminations Ex1and Ex2 is allowed to pass through a corresponding one of the slitportions.

The primary image is irradiated onto the sample S on the stage 20through the observation optical system 40. The observation opticalsystem 40 includes a condenser lens 41, the dichroic mirror 42, adichroic mirror 43, the objective 44, the bandpass filter 45, and acondenser lens 46. The line illuminations Ex1 and Ex2 are collimated bythe condenser lens 41 paired with the objective 44, reflected off thedichroic mirrors 42 and 43 to pass through the objective 44, andirradiated onto the sample S.

Illuminations as illustrated in FIG. 4 are formed on the surface of thesample S. Fluorescence excited with each of the illuminations iscollected by the objective 44, is reflected off the dichroic mirror 43,passes through the dichroic mirror 42 and the bandpass filter 45 used toblock excitation light, and is collected again by the condenser lens 46to enter the spectroscopic imaging section 30.

The spectroscopic imaging section 30 includes the observation slit 31,the imaging device 32 (32 a, 32 b), a first prism 33, mirrors 34,diffraction gratings 35 (wavelength dispersion elements), and a secondprism 36.

The observation slit 31 is arranged with respect to a light-collectingpoint of the condenser lens 46, and includes the same number of slitportions as the number of excitation lines. Fluorescence spectra fromthe two excitation lines having passed through the observation slit 31are separated by the first prism 33, and each reflected off the gratingsurface of the diffraction grating 35 through the mirror 34 to befurther separated into fluorescence spectra of respective excitationwavelengths. The four fluorescence spectra obtained by performing theseparation described above enter the imaging devices 32 a, 32 b throughthe mirrors 34 and the second prism 36, and are provided as information(x, λ) that is spectroscopic data.

The pixel size (nm/pixel) of each of the imaging devices 32 a, 32 b isnot particularly limited, and is set to, for example, between 2 nm and20 nm, inclusive. The variance may be provided using a pitch of thediffraction grating 35 or may be optically provided, or may be providedusing hardware binning of each of the imaging devices 32 a, 32 b.

The stage 20 and the scanning mechanism 50 form the XY-stage, and movethe sample S in the X-axis direction and in the Y-axis direction inorder to acquire a fluorescence image of the sample S. In whole slideimaging (WSI), an operation is repeatedly performed, the operationincluding scanning the sample S in the Y-axis direction, moving thesample S in the X-axis direction, and further scanning the sample S inthe Y-axis direction (refer to FIG. 8 ).

The non-fluorescence observation section 70 includes, for example, alight source 71, the dichroic mirror 43, the objective 44, a condenserlens 72, and an imaging device 73. FIG. 2 illustrates an observationsystem using dark field illumination as the non-fluorescence observationsystem.

The light source 71 is arranged below the stage 20, and irradiatesillumination light onto the sample S on the stage 20 from a sideopposite to the line illuminations Ex1 and Ex2. In the case of the darkfield illumination, the light source 71 performs irradiation from theoutside of a numerical aperture (NA) of the objective 44, and an imageof the light being diffracted by the sample S (a dark field image) andpassing through the objective 44, the dichroic mirror 43, and thecondenser lens 72 is captured using the imaging device 73. The use ofthe dark field illumination makes it possible to observe even anapparently transparent sample such as a fluorescently stained samplewith contrast.

Note that this dark field image may be observed simultaneously withfluorescence, and may be used for real-time focusing. In this case, awavelength that does not affect a fluorescence observation may beselected as an illumination wavelength. The non-fluorescence observationsection 70 is not limited to being formed of an observation system thatacquires a dark field image, and may be formed of an observation systemthat is capable of acquiring a non-fluorescence image such as a brightfield image, a phase contrast image, a phase image, or anin-line-hologram image. For example, various observation methods such asa schlieren method, a phase contrast method, polarized light microscopy,and an epi-illumination method may be adopted as a method for acquiringa non-fluorescence image. The illumination light source is not limitedto being situated below the stage, and it may be situated above thestage or around the objective. Further, not only a method for performingfocusing control in real time, but also another method such as apre-focusing map method that records a focus coordinate (Z coordinate)in advance may be adopted.

FIG. 11 schematically illustrates another configuration example of thespectroscopic imaging section. A spectroscopic imaging section 130illustrated in the figure includes a single imaging device 32. Eachpiece of fluorescence having passed through the observation slit 31including a slit portion passes through a relay optical system (thefirst prism 33, the mirror 34, and a mirror 37) and a wavelengthdispersion element (such as a prism) 38 that is arranged on the way, andan image of the piece of fluorescence is formed on the imaging device 32to be provided as data (x, λ) (refer to FIG. 5 ). Here, the number ofslit portions is the same as the number of excitation lines. In thiscase, a value on a pixel basis into which the excitation-light intervalΔy has been converted is determined such that dispersed spectra do notoverlap on the imaging device 32.

[Processing Unit]

Fluorescence spectra acquired by the imaging device 32 (32 a, 32 b) areoutput to the processing unit 2. The processing unit 2 includes thestorage 21, and further includes a data calibration section 22 thatcalibrates spectroscopic data stored in the storage 21, and an imageformation section 23 that forms a fluorescence image of the sample S onthe basis of the spectroscopic data and the interval Δy between the lineilluminations Ex1 and Ex2.

FIG. 12 is a flowchart illustrating an example of a procedure ofprocessing performed in the processing unit 2.

The storage 21 stores therein spectroscopic data (the fluorescencespectra Fs1 and Fs2 (refer to FIGS. 5 and 7 )) acquired by thespectroscopic imaging section 30 (Step 101). A standard spectrum ofautofluorescence related to the sample S and a standard spectrum of onlya dye are stored in the storage 21 in advance.

The storage 21 only extracts a wavelength region of interest from apixel array of the imaging device 32 in the wavelength direction toimprove the recording frame rate. The wavelength region of interestcorresponds to, for example, a range of visible light (380 nm to 780 nm)or a wavelength range determined by an emission wavelength of a dye thatstains the sample S.

Examples of a wavelength region other than the wavelength region ofinterest include a sensor region in which there exists light ofunnecessary wavelength, a sensor region in which there obviously existsno signal, and a region of an excitation wavelength to be blocked by thedichroic mirror 42 or the bandpass filter 45 in a light path on the way.Further, the wavelength region of interest above the sensor may beswitched according to a state of a line illumination. For example, whenthere are only a few excitation wavelengths used for a lineillumination, the wavelength region above the sensor is also limited,and the frame rate becomes higher according to the limitation.

The data calibration section 22 performs calibration includingconverting the spectroscopic data stored in the storage 21 into data ona wavelength basis from pixel data (x, λ) such that all of the pieces ofspectral data are complemented such that all of the pieces of spectraldata are pieces of data in a unit of wavelength (such as [nm] or [μm])and have discrete values in common, and such that the complementedpieces of spectral data are output (Step 102).

The pixel data (x, λ) is not limited to being well arranged in a pixelcolumn of the imaging device 32, and may be distorted due to a slighttilt or a distortion of an optical system. Thus, when, for example, thedata is converted into a unit of wavelength from a unit of pixel using alight source of a known wavelength, conversion into differentwavelengths (nm values) is performed with respect to all of the xcoordinates. Since it is difficult to deal with data in this state,conversion of the data into data including arranged integers isperformed by interpolation (such as linear interpolation or splineinterpolation) (Step 102).

Further, there is a possibility that sensitivity will become nonuniformin a long-axis direction of a line illumination (the X-axis direction).The sensitivity becomes nonuniform due to nonuniform illumination or avariation in slit width, and this results in uneven brightness of acaptured image. Thus, in order to eliminate such nonuniformity, the datacalibration section 22 makes the sensitivity uniform using an arbitrarylight source and its representative spectrum (such as an averagespectrum and spectral radiance of the light source), and outputs theuniform sensitivity (Step 103). An instrumental error is eliminated bymaking the sensitivity uniform, and this makes it possible to save theeffort to measure an individual component spectrum every time uponanalyzing a waveform of a spectrum. Furthermore, it is also possible tooutput an approximate quantitative value of the number of fluorescencedyes using a brightness value in which the sensitivity has beencalibrated.

When the spectral radiance [W/(sr·m²·nm)] is adopted to the calibratedspectrum, sensitivities of the imaging device 32 that correspond torespective wavelengths are also corrected. As described above, byperforming calibration such that adjustment to a spectrum used as areference is performed, it is no longer necessary to measure, for eachapparatus, a reference spectrum used to perform calculation for colorseparation. It is possible to utilize, for a stable dye in the same lot,data obtained in a single image-capturing. Further, when thefluorescence spectra intensity per dye molecule is provided in advance,it is possible to output an approximate value of the number offluorescence dye molecules, the approximate value being a value intowhich a brightness value in which the sensitivity has been calibrated,has been converted. This value is highly quantitative since anautofluorescence component has also been separated.

The processing described above is similarly performed with respect toranges of illuminations performed with the line illuminations Ex1 andEx2, the ranges of illuminations being situated in the sample S scannedin the Y-axis direction. Accordingly, spectroscopic data (x, y, λ) ofeach fluorescence spectrum is obtained for an entire range of the sampleS. The obtained spectroscopic data (x, y, λ) is stored in the storage21.

The image formation section 23 forms a fluorescence image of the sampleS on the basis of the spectroscopic data stored in the storage 21 (orthe spectroscopic data calibrated by the data calibration section 22),and on the basis of an interval corresponding to an inter-axis distance(Δy) of the excitation lines Ex1 and Ex2 (Step 104). In the presentembodiment, the image formation section 23 forms, as a fluorescenceimage, an image in which coordinates detected by the imaging device 32have been corrected using a value corresponding to the interval (Δy)between the plurality of line illuminations Ex1 and Ex2.

Three-dimensional data based on one of the line illuminations Ex1 andEx2 is data obtained by shifting the coordinate by Δy from thecoordinate of another of the line illuminations Ex1 and Ex2 with respectto the Y-axis. Thus, correction is performed on the basis of a value ofΔy that is recorded in advance, or on the basis of a value of Δy that iscalculated from output of the imaging device 32, and data obtained bythe correction is output. Here, correction is performed with respect toa difference in coordinates detected by the imaging device 32 such thatthe pieces of three-dimensional data based on the respective lineilluminations Ex1 and Ex2 are pieces of data on the same coordinates.

The image formation section 23 performs processing (stitching) forconnecting captured images to obtain one large image (WSI) (Step 105).This makes it possible to acquire a multiplexed pathological imagerelated to the sample S (the observation target Sa). The formedfluorescence image is output to the display section 3 (Step 106).

Further, on the basis of a standard spectrum of autofluorescence of thesample S and a standard spectrum of only a dye of the sample S that arestored in the storage 21 in advance, the image formation section 23performs separation calculation of a component distribution ofautofluorescence and a dye of the sample S from spectroscopic data(measurement spectrum) obtained by performing image-capturing. It ispossible to adopt least squares, weighted least squares, or the like asa calculation method, and a coefficient is calculated such that thespectroscopic data obtained by performing image-capturing is a linearsum of the standard spectra described above. A distribution ofcalculated coefficients is stored in the storage 21, and is output to bedisplayed on the display section 3 in the form of an image (Steps 107and 108).

[Conclusion]

As described above, the present embodiment makes it possible to providea multiple fluorescence scanner in which the image-capturing time is notincreased even when there is an increase in the number ofobservation-target dyes.

In other words, data captured by the multiple fluorescence scanner isthree-dimensional data (x, y, λ). Thus, in the case of performing planarimage-capturing, it is not possible to acquire all of the pieces of data(x, y, λ) at a time, and thus there is a need to chronologically switchA to perform image-capturing. Further, when irradiation is performedusing a plane light source (x, y) to perform excitation, a method forchronologically switching the excitation wavelength is indispensable dueto physical constraints. In order to solve these problems, in thepresent embodiment, fluorescence is excited with a plurality of lineilluminations of different wavelengths, the plurality of lineilluminations being a plurality of line illuminations arranged parallelto each other on different axes. Each linearly excited fluorescencespectrum is separated into (x, λ), and image-capturing is performed at atime using a two-dimensional sensor (an imaging device). Thisconfiguration makes it possible to separate the excitation wavelengthspatially and not temporally. Thus, the image-capturing time is notincreased even when there is an increase in the number ofobservation-target dyes.

There is a need for one-dimensional scanning (Y direction) to acquiretwo-dimensional data in this method. Thus, apparently it takes time toperform image-capturing, compared to when a plane excitation isperformed. However, if the same light source emission power as that usedfor a plane excitation is allowed to be used for a line irradiation, itwill be possible to excite fluorescence with brighter light since theline irradiation achieves a high power density due to its small area.The reason is that the fluorescence intensity is linearly increased withrespect to the excitation power density until the energy absorption of adye is saturated due to the characteristics of fluorescence. Thisresults in a reduction in exposure time, and thus it is possible toperform image-capturing fastest in principle to obtain (x, y, λ) (sincethe excited fluorescence is not discarded, a high energy efficiency isachieved, and this makes it possible to acquire data at high speed).Further, due to scanning being performed at a constant speed, thismethod is advantageous in that it is superior to a plane excitationmethod in large-area image-capturing, the plane excitation method beinga method in which there is a need to repeatedly perform stop-and-goprocessing.

Further, the present embodiment makes it possible to performimage-capturing on a fluorescence dye with a higher degree of energyefficiency, compared to using a plane-excitation spectroscopic imagingapparatus of a time-division type. Furthermore, it is no longernecessary to measure, for each apparatus, a reference spectrum used toperform calculation for color separation. Thus, it is possible toutilize, for a stable dye in the same lot in a next measurement, dataobtained in a single image-capturing. Moreover, the quantitativemeasurement of a dye molecule makes it possible to quantitativelyevaluate the number of antigens in a tissue or on a cell surface.

[Display Section]

Next, the display section 3 is described.

FIG. 13 is a diagram describing a screen of the display section 3. Thedisplay section 3 may be formed of a monitor integrally attached to theprocessing unit 2, or may be a display apparatus connected to theprocessing unit 2. The display section 3 includes a display element suchas a liquid crystal device or an organic EL device, and a touch sensor,and is configured as a user interface (UI) that displays a setting forinputting an image-capturing condition, a captured image, and the like.

As illustrated in FIG. 13 , the display section 3 includes a primaryscreen 301, a thumbnail-image display screen 302, a slide-informationdisplay screen 303, and a display screen 304 for a list of slides forwhich image-capturing has been completed. The primary screen 301includes a display region 305 for an operation button (a key) or thelike used for image-capturing, a setting region 306 for an excitationlaser (the excitation section 10), detection setting regions 307 and 308for fluorescence spectra from the line illuminations Ex1 and Ex2, andthe like. It is sufficient if there constantly exists at least one ofthese display regions 305 to 308, and one of the display regions mayinclude another of the display regions.

The fluorescence observation apparatus 100 sequentially performs takeoutof a slide (the sample S) from a slide rack (not illustrated), read ofslide information, capturing of a thumbnail image of the slide, settingof an exposure time, and the like. The slide information includespatient information, information regarding a tissue site, a disease, andstaining, and the like, and is read from a bar code or the QR code(registered trademark) attached to the slide. The thumbnail image andthe slide information of the sample S are respectively displayed on thedisplay screens 302 and 303. Information regarding a slide on whichimage-capturing has been completed is displayed on the screen 304 in theform of a list.

In addition to a fluorescence image of the sample S, an image-capturingstate for a slide on which image-capturing is currently being performedis displayed on the primary screen 301. An excitation laser (the lineilluminations Ex1 and Ex2) is displayed or set in the setting region306, and a fluorescence spectrum from the excitation laser is displayedor set in the detection setting regions 307 and 308.

FIG. 14 illustrates an example of a screen configuration of the settingregion 306 for an excitation laser. Here, ON/OFF of output of each ofthe excitation light sources L1 to L4 is selected and switched by atouch operation being performed on a checkbox 81. Further, the magnitudeof the output of each light source is set through an operation section82. In this example, the line illumination Ext is set at a singlewavelength of the excitation light source L1.

FIG. 15 illustrates an example of a screen configuration of thedetection setting region 307 for a fluorescence spectrum from the lineillumination Ext. FIG. 16 illustrates an example of a screenconfiguration of the detection setting region 308 for a fluorescencespectrum from the line illumination Ex2. The vertical axis representsbrightness, and the horizontal axis represents a wavelength.

In FIGS. 15 and 16 , an index 83 indicates that the excitation lightsources (L1, L2, and L4) are on, and a longer length of the indicator 83indicates a greater power of a light source. The detection wavelengthrange for a fluorescence spectrum 85 is set with a setting bar 84.

The method for displaying the fluorescence spectrum 85 is notparticularly limited, and, for example, an average spectrum of all ofthe pixels of the imaging device 32 (wavelength×intensity) is displayedto be the fluorescence spectrum 85. It is possible to set thefluorescence spectrum 85 according to the wavelength and the power of anexcitation light source. The fluorescence spectrum 85 is displayed inthe form of a current average, or in the form of a waveform, thewaveform being calculated from a waveform of which an image is mostpreviously captured, the calculation being performed in consideration ofa change in the setting.

Further, as illustrated in FIGS. 15 and 16 , the fluorescence spectrum85 may be displayed by a heat map method in which frequency informationregarding a value is indicated with light and dark. In this case, it isalso possible to visualize a signal variance that is not realized usingan average.

Note that the vertical axis of a graph used to display the fluorescencespectrum 85 is not limited to a linear axis, and may be a logarithmicaxis or a hybrid axis (a biexponential axis).

The display section 3 is capable of displaying a fluorescence spectrumseparately for each excitation line (Ex1, Ex2). Further, the displaysection 3 further includes a UI that includes an operation region usedto explicitly display a wavelength and a power of a light source, thewavelength being irradiated onto each excitation line. The displaysection 3 further includes a UI used to display a detection wavelengthrange for each fluorescence spectrum. In other words, the displaysection 3 is configured such that a reading region of the imaging device32 is changed on the basis of the set wavelength range.

This makes it possible to present an image-capturing condition to a userin an easy-to-understand manner in a fluorescence observation apparatusin which excitation is performed on different axes. In particular, byproviding the detection setting regions 307 and 308 for fluorescencespectra to the display section 3, it is possible to display, in aneasy-to-understand manner, a relationship between an excitation line andan excitation wavelength, and a relationship between an excitationwavelength and an image-capturing wavelength range even when excitationis performed on different axes.

The display section 3 displays, on the primary screen 301, afluorescence image of the sample S that is output from the imageformation section 23. The fluorescence image output from the imageformation section 23 to the display section 3 is presented to the userin a state in which correction has been performed using a value (theinterval Δy between the line illuminations Ex1 and Ex2) that correspondsto a difference in detected coordinates between slits on different axes(the respective slit portions of the observation slit 31). This enablesthe user to recognize an image obtained by multiply-displaying pieces ofdecomposition image data without being aware of a difference betweendetected positions on different axes.

For example, as illustrated in FIG. 17 , a plurality of decompositionimages (an image related to a dye 1 and an image related to a dye 2) isgenerated using pieces of spectral data based on the plurality of lineilluminations Ex1 and Ex2, and the respective images of different colorsare superimposed to be displayed on the primary screen 301. Here, theimage related to the dye 1 is superimposed on the image related to thedye 2 by performing correction with respect to a difference in Ycoordinate that corresponds to Δy.

Each decomposition image corresponds to a standard spectrum used forseparation calculation, that is, a staining dye. In addition to a dyeimage in which respective decomposition images are superimposed, ascreen for selecting a displayed dye may be displayed on the primaryscreen 301. In this case, the image display is switched in conjunctionwith selection of a dye, and only images corresponding to the dyes 1 and2 are displayed when the dyes 1 and 2 are selected, as illustrated inFIG. 17 .

The correction value Δy described above is stored in the storage 21 andmanaged as internal information. The display section 3 may be capable ofdisplaying information regarding Δy, or may be capable of changing thedisplayed Δy. The correction value (Δy) may include not only a valueused to perform correction with respect to a distance between slits (oran interval between line illuminations), but also a value used toperform correction with respect to an amount of distortion such asdistortion in an optical system. When a spectrum of each dye is detectedusing a different camera (an imaging device), the correction value (Δy)may include a correction amount related to detected coordinates in theY-axis direction with respect to each camera.

<Modification>

In the embodiment described above, the processing of adjusting an imageusing Δy (S104 in FIG. 12 ) and the stitching processing (S105 in thesame figure) are respectively performed in the processing unit 2 beforethe processing of calculation for color separation (S107 in the samefigure). However, the processing procedure is not limited to this. Thisprocedure has the advantage that not only a color-separation image butalso stitching data of spectral data (spectroscopic data) is obtained.On the other hand, a huge amount of data is stored in the storage 21.Thus, at least one of the processing of adjusting an image using Δy orthe stitching processing may be performed after the processing ofcalculation for color separation, as illustrated in FIGS. 18 and 19 .

FIG. 18 illustrates an example of the processing procedure in which thestitching processing (S105) is performed after the processing ofcalculation for color separation (S107). In this example, stitching isnot performed with respect to spectral data having a large data size,but stitching is performed only with respect to an image in whichcalculation for color separation has been performed. In this case, theprocessing of adjusting an image using Δy (S104) is performed withrespect to the spectral data before calculation is performed for colorseparation. This example makes it possible to improve the SN ratio of aseparation image by performing color separation with respect to dataobtained by connecting pieces of spectral data of a plurality ofexcitation lines.

On the other hand, FIG. 19 illustrates an example of the processingprocedure in which the processing of adjusting an image using Δy (S104)and the stitching processing (S105) are performed after the processingof calculation for color separation (S107). In this example, the SNratio of a separation image is not expected to be improved sincestitching is not performed with respect to spectral data. However, it ispossible to perform a higher level of alignment correction such asalignment correction in consideration of a subpixel, since theprocessing of adjusting an image using Δy is performed with respect to aseparation image with a small amount of data.

Next, a modification of the configuration of the fluorescenceobservation apparatus 100 described above is described.

FIG. 20 is a schematic block diagram of a fluorescence observationapparatus 101 according to a first modification, and FIG. 21 is aschematic block diagram of a fluorescence observation apparatus 102according to a second modification. The fluorescence observationapparatuses 101 and 102 each include the observation unit 1, theprocessing unit 2, the display section 3, and a control program 81.

The control program 81 is a program that causes the fluorescenceobservation apparatuses 101 and 102 to perform the same function as acontrol function performed by the controller 80 of the fluorescenceobservation apparatus 100 described above. In the fluorescenceobservation apparatus 101 illustrated in FIG. 20 , the control program81 is provided in a state of being stored in a recording medium such asa magnetic disk, an optical disk, a magneto-optical disk, or a flashmemory, and is downloaded to and used by a computer C or the like thatis connected to the fluorescence observation apparatus 101.

On the other hand, in the fluorescence observation apparatus 102illustrated in FIG. 21 , the control program 81 distributed from theoutside through a network such as the Internet is downloaded to and usedby the computer C or the like. In this case, the fluorescenceobservation apparatus 102 and a code used to acquire the control program81 are packaged to be provided.

The electronic computer C to which the control program 81 has beendownloaded acquires various data used to control the excitation section10, the spectroscopic imaging section 30, the scanning mechanism 50, thefocusing mechanism 60, the non-fluorescence observation section 70, andthe like. A control algorithm of the downloaded control program 81 isexecuted, and control conditions for the fluorescence observationapparatus 101, 102 are calculated. Conditions for the fluorescenceobservation apparatus 101, 102 are automatically controlled by thecomputer C giving an instruction to the fluorescence observationapparatus 101, 102 on the basis of the calculated conditions.

Note that the present technology may also take the followingconfigurations.

(1) A fluorescence observation apparatus, including:

a stage that is capable of supporting a fluorescently stainedpathological specimen;

an excitation section that irradiates the pathological specimen on thestage with a plurality of line illuminations of different wavelengths,the plurality of line illuminations being a plurality of lineilluminations situated on different axes and parallel to a certain-axisdirection; and

a spectroscopic imaging section that includes at least one imagingdevice capable of separately receiving pieces of fluorescencerespectively excited with the plurality of line illuminations.

(2) The fluorescence observation apparatus according to (1), in which

the excitation section is configured to irradiate, onto the pathologicalspecimen and as the plurality of line illuminations, a plurality of lineilluminations each having different wavelengths in combination.

(3) The fluorescence observation apparatus according to (2), in which

the spectroscopic imaging section further includes a wavelengthdispersion element that separates each of the pieces of fluorescencerespectively excited with the plurality of line illuminations.

(4) The fluorescence observation apparatus according to any one of (1)to (3), in which

the spectroscopic imaging section further includes an observation slitincluding a plurality of slit portions, each of the pieces offluorescence respectively excited with the plurality of lineilluminations being allowed to pass through a corresponding one of theplurality of slit portions.

(5) The fluorescence observation apparatus according to any one of (1)to (5), further including

a scanning mechanism that scans the plurality of line illuminations overthe stage in a direction orthogonal to the certain-axis direction.

(6) The fluorescence observation apparatus according to any one of (1)to (5), further including

a processing unit that includes a storage that stores thereinspectroscopic data that indicates a correlation between a wavelength ofeach of the plurality of line illuminations and fluorescence received bythe imaging device.

(7) The fluorescence observation apparatus according to (6), in which

the processing unit further includes an image formation section thatforms a fluorescence image of the pathological specimen on the basis ofthe spectroscopic data stored in the storage and an interval between theplurality of line illuminations.

(8) The fluorescence observation apparatus according to (7), in which

the image formation section forms, as the fluorescence image, an imagein which coordinates detected by the imaging device have been correctedusing a value corresponding to the interval between the plurality ofline illuminations.

(9) The fluorescence observation apparatus according to (6), in which

the processing unit further includes a data calibration section thatcalibrates spectroscopic data stored in the storage.

(10) The fluorescence observation apparatus according to any one of (7)to (9), in which

the storage stores therein standard spectra in advance, the standardspectra being a standard spectrum of autofluorescence related to thepathological specimen and a standard spectrum of only a dye that stainsthe pathological specimen, and

the image formation section outputs a component distribution of thespectroscopic data on the basis of the standard spectrum of theautofluorescence and the standard spectrum of only the dye.

(11) The fluorescence observation apparatus according to (4), in which

the imaging device includes a plurality of imaging devices each capableof receiving the fluorescence passing through the observation slit.

(12) The fluorescence observation apparatus according to any one of (1)to (11), further including:

a non-fluorescence observation section that includes a light source thatilluminates the pathological specimen on the stage; and

an imaging section that acquires a non-fluorescence image of thepathological specimen.

(13) The fluorescence observation apparatus according to any one of (1)to (12), further including:

a display section that displays fluorescence spectra separately for eachof the plurality of line illuminations, the fluorescence spectra beingrespectively excited with the plurality of line illuminations.

(14) The fluorescence observation apparatus according to (13), in which

the display section includes an operation region in which a wavelengthand output of each of the plurality of line illuminations are allowed tobe set.

(15) The fluorescence observation apparatus according to (13) or (14),in which

the display section includes a display region used to display adetection wavelength range for the fluorescence spectrum.

(16) A fluorescence observation method, including:

irradiating a pathological specimen on a stage with a plurality of lineilluminations of different wavelengths, the plurality of lineilluminations being a plurality of line illuminations situated ondifferent axes and parallel to a certain-axis direction; and

separately receiving pieces of fluorescence respectively excited withthe plurality of line illuminations.

(17) The fluorescence observation method according to (16), furtherincluding

scanning the plurality of line illuminations over the stage in adirection orthogonal to the certain-axis direction.

(18) The fluorescence observation method according to (16) or (17), inwhich

a plurality of line illuminations each having different wavelengths incombination is used as the plurality of line illuminations.

REFERENCE SIGNS LIST

-   1 observation unit-   2 processing unit-   3 display section-   10 excitation section-   20 stage-   21 storage-   22 data calibration section-   23 image formation section-   30, 130 spectroscopic imaging section-   31 observation slit-   32, 32 a, 32 b imaging device-   35 diffraction grating-   38 prism-   50 scanning mechanism-   70 non-fluorescence observation section-   80 controller-   81 control program-   100,101,102 fluorescence observation apparatus-   Ex1, Ex2 line illumination-   S sample

The invention claimed is:
 1. A fluorescence observation apparatus,comprising: a stage that is capable of supporting a fluorescentlystained pathological specimen; an excitation section that irradiates thepathological specimen on the stage with a plurality of lineilluminations of different wavelengths, the plurality of lineilluminations being a plurality of line illuminations situated ondifferent axes and being collimated by a collimator to a certain-axisdirection; and a spectroscopic imaging section that includes at leastone imaging device capable of separately receiving pieces offluorescence respectively excited with the plurality of lineilluminations, wherein optical axes of the plurality of lineilluminations are parallel between an objective lens and thepathological specimen and wherein optical axes of theseparately-received pieces of fluorescence are parallel when incident onthe spectroscopic imaging section.
 2. The fluorescence observationapparatus according to claim 1, wherein the excitation section isconfigured to irradiate, onto the pathological specimen and as theplurality of line illuminations, a plurality of line illuminations eachhaving different wavelengths in combination.
 3. The fluorescenceobservation apparatus according to claim 2, wherein the spectroscopicimaging section further includes a wavelength dispersion element thatseparates each of the pieces of fluorescence respectively excited withthe plurality of line illuminations.
 4. The fluorescence observationapparatus according to claim 1, wherein the spectroscopic imagingsection further includes an observation slit including a plurality ofslit portions, each of the pieces of fluorescence respectively excitedwith the plurality of line illuminations being allowed to pass through acorresponding one of the plurality of slit portions.
 5. The fluorescenceobservation apparatus according to claim 1, further comprising ascanning mechanism that scans the plurality of line illuminations overthe stage in a direction orthogonal to the certain-axis direction. 6.The fluorescence observation apparatus according to claim 1, furthercomprising a processing unit that includes a storage that stores thereinspectroscopic data that indicates a correlation between a wavelength ofeach of the plurality of line illuminations and fluorescence received bythe imaging device.
 7. The fluorescence observation apparatus accordingto claim 6, wherein the processing unit further includes an imageformation section that forms a fluorescence image of the pathologicalspecimen on a basis of the spectroscopic data stored in the storage andan interval between the plurality of line illuminations.
 8. Thefluorescence observation apparatus according to claim 7, wherein theimage formation section forms, as the fluorescence image, an image inwhich coordinates detected by the imaging device have been correctedusing a value corresponding to the interval between the plurality ofline illuminations.
 9. The fluorescence observation apparatus accordingto claim 6, wherein the processing unit further includes a datacalibration section that calibrates spectroscopic data stored in thestorage.
 10. The fluorescence observation apparatus according to claim7, wherein the storage stores therein standard spectra in advance, thestandard spectra being a standard spectrum of autofluorescence relatedto the pathological specimen and a standard spectrum of only a dye thatstains the pathological specimen, and the image formation sectionoutputs a component distribution of the spectroscopic data on a basis ofthe standard spectrum of the autofluorescence and the standard spectrumof only the dye.
 11. The fluorescence observation apparatus according toclaim 4, wherein the imaging device includes a plurality of imagingdevices each capable of receiving the fluorescence passing through theobservation slit.
 12. The fluorescence observation apparatus accordingto claim 1, further comprising: a non-fluorescence observation sectionthat includes a light source that illuminates the pathological specimenon the stage; and an imaging section that acquires a non-fluorescenceimage of the pathological specimen.
 13. The fluorescence observationapparatus according to claim 1, further comprising: a display sectionthat displays fluorescence spectra separately for each of the pluralityof line illuminations, the fluorescence spectra being respectivelyexcited with the plurality of line illuminations.
 14. The fluorescenceobservation apparatus according to claim 13, wherein the display sectionincludes an operation region in which a wavelength and output of each ofthe plurality of line illuminations are allowed to be set.
 15. Thefluorescence observation apparatus according to claim 13, wherein thedisplay section includes a display region used to display a detectionwavelength range for the fluorescence spectrum.
 16. A fluorescenceobservation method, comprising: irradiating a pathological specimen on astage with a plurality of line illuminations of different wavelengths,the plurality of line illuminations being a plurality of lineilluminations situated on different axes and being collimated by acollimator to a certain-axis direction; and separately receiving piecesof fluorescence respectively excited with the plurality of lineilluminations, wherein optical axes of the plurality of lineilluminations are parallel between an objective lens and thepathological specimen and wherein optical axes of theseparately-received pieces of fluorescence are parallel when incident ona spectroscopic imaging section.
 17. The fluorescence observation methodaccording to claim 16, further comprising scanning the plurality of lineilluminations over the stage in a direction orthogonal to thecertain-axis direction.
 18. The fluorescence observation methodaccording to claim 16, wherein a plurality of line illuminations eachhaving different wavelengths in combination is used as the plurality ofline illuminations.